Digital imprinting of RNA recognition and processing on a self-assembled nucleic acid matrix.

Redhu SK, Castronovo M, Nicholson AW - Sci Rep (2013)

Bottom Line:
The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment.The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input.These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

ABSTRACTThe accelerating progress of research in nanomedicine and nanobiotechnology has included initiatives to develop highly-sensitive, high-throughput methods to detect biomarkers at the single-cell level. Current sensing approaches, however, typically involve integrative instrumentation that necessarily must balance sensitivity with rapidity in optimizing biomarker detection quality. We show here that laterally-confined, self-assembled monolayers of a short, double-stranded(ds)[RNA-DNA] chimera enable permanent digital detection of dsRNA-specific inputs. The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input. These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

f3: Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).

Mentions:
The height of a dsDNA monolayer is proportional to its density2122. The question thus arises whether the outputs observed in this study also exhibit a similar dependence on monolayer density. Fig. 3a shows the output from Input 1 as a function of ds[RNA-DNA] matrix density, as qualitatively gauged by initial height (HIN). Specifically, an HIN of >11.5 nm corresponds to the maximum height calculated for a near-vertical orientation of the molecules (see also Discussion), reflecting the near-maximal density of the ds[RNA-DNA] matrix. Output 1 (Fig. 3a, solid red triangles) shows that the change in height of the ds[RNA-DNA] matrix linearly correlates with HIN, indicating that the RNase III cleavage site remains accessible, regardless of density. When BamHI is included, the results (Output 1+; Fig. 3a, open triangles) show that the dsDNA segment is accessible to BamHI in the presence of RNase III, also regardless of density. A requirement for the prior action of RNase III for the BamHI reaction is likely, since the same results are obtained if the ds[RNA-DNA] matrix is pre-treated with RNase III, then incubated with BamHI following removal of RNase III (data not shown). In contrast, the action of BamHI is inefficient when the relatively dense ds[RNA-DNA] matrix (HIN > 10 nm) does not receive prior treatment with RNase III (Output 3+, Fig. 2d; open diamonds). Finally, BamHI action is fully suppressed in the presence of the E110A mutant, regardless of matrix density (Output 2+, open circles). The results shown in Figs. 2 and 3 indicate that BamHI action exhibits a dependence on RNase III catalytic action (+RNase III) or RNase III binding (+E110A) that occurs in the dsRNA segment. Thus, BamHI can effectively capture either a catalytic or noncatalytic RNA-protein interaction by generating a specific, permanent, AFM-readable matrix imprint.

f3: Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).

Mentions:
The height of a dsDNA monolayer is proportional to its density2122. The question thus arises whether the outputs observed in this study also exhibit a similar dependence on monolayer density. Fig. 3a shows the output from Input 1 as a function of ds[RNA-DNA] matrix density, as qualitatively gauged by initial height (HIN). Specifically, an HIN of >11.5 nm corresponds to the maximum height calculated for a near-vertical orientation of the molecules (see also Discussion), reflecting the near-maximal density of the ds[RNA-DNA] matrix. Output 1 (Fig. 3a, solid red triangles) shows that the change in height of the ds[RNA-DNA] matrix linearly correlates with HIN, indicating that the RNase III cleavage site remains accessible, regardless of density. When BamHI is included, the results (Output 1+; Fig. 3a, open triangles) show that the dsDNA segment is accessible to BamHI in the presence of RNase III, also regardless of density. A requirement for the prior action of RNase III for the BamHI reaction is likely, since the same results are obtained if the ds[RNA-DNA] matrix is pre-treated with RNase III, then incubated with BamHI following removal of RNase III (data not shown). In contrast, the action of BamHI is inefficient when the relatively dense ds[RNA-DNA] matrix (HIN > 10 nm) does not receive prior treatment with RNase III (Output 3+, Fig. 2d; open diamonds). Finally, BamHI action is fully suppressed in the presence of the E110A mutant, regardless of matrix density (Output 2+, open circles). The results shown in Figs. 2 and 3 indicate that BamHI action exhibits a dependence on RNase III catalytic action (+RNase III) or RNase III binding (+E110A) that occurs in the dsRNA segment. Thus, BamHI can effectively capture either a catalytic or noncatalytic RNA-protein interaction by generating a specific, permanent, AFM-readable matrix imprint.

Bottom Line:
The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment.The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input.These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

ABSTRACTThe accelerating progress of research in nanomedicine and nanobiotechnology has included initiatives to develop highly-sensitive, high-throughput methods to detect biomarkers at the single-cell level. Current sensing approaches, however, typically involve integrative instrumentation that necessarily must balance sensitivity with rapidity in optimizing biomarker detection quality. We show here that laterally-confined, self-assembled monolayers of a short, double-stranded(ds)[RNA-DNA] chimera enable permanent digital detection of dsRNA-specific inputs. The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input. These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.